Myocyte function is depressed in heart failure. We have found marked alterations in steady state maximum force development, myofilament Ca2+ sensitivity, and cross-bridge cycling rate at end-stage congestive heart failure in a rat model. We have also obtained evidence for maladapted contractile protein phosphorylation in heart failure that are functionally relevant. We now extend our studies to a) determine which contractile sub- proteins are the key players in the decline of myocyte function during the development of congestive heart failure using 3 distinct models of cardiac failure in the rat (myocardial infarction, pressure overload, volume overload), and b) elucidate the impact of heart failure and contractile protein phosphorylationon myofilament activation/relaxation dynamics. Overall, we will test the hypothesisthat up-regulation of protein kinase C and specific phosphorylation of sarcomeric targets leads to sarcomere maladaptationanddecompensation. (1) What are the cellular mechanisms of decreased myofilament force and Ca2+ sensitivity in congestive heart failure. Studies in this aim are designed to reveal 1) whether alterations in hemodynamic parameters in the intact animal correlate with altered steady state myofilament function during the development of failure, 2) which myofilament proteins are responsible for the observed depressed function, and, 3) determine whether and/or to what extent myofilament protein phosphorylation is involved. Myofilament function (tension development, Ca2+ sensitivity, ATP turnover) will be studied in isolated skinned trabeculae and isolated single skinned myocytes. The functional role of contractile protein subunit isoform distribution/phosphorylation status will be studied by a) determination of proteins involved by proteomic analysis of skinned myocardium;b) protein exchange in skinned fibers with recombinant protein as well as purified troponin from control or failing hearts. (2) Altered myofilament activation/relaxation kinetics: Extent and molecular mechanisms in CHF? Steady force-Ca2" relationships reveal but a limited aspect of myofilament function. Studies in this aim will utilize single myofibril techniques to measure the dynamic relation between [Ca2+] and myofilament force. Specifically, we will 1) determine the extent of activation/relaxation acto-myosin kinetics abnormalities during the development of cardiac failure in the rat models 2) correlate these dynamic myofilament properties to in- vivo hemodynamics, 3) determine the impact of contractile protein isoform distribution/phosphorylation status on activation/relaxation kinetics in healthy, non-diseased, single skinned cardiac myofibrils via contractile sub- protein exchange, 4) determine whether contractile sub-protein exchange can reverse activation/relaxation single myofibril kinetic dysfunction in the 3 experimental cardiac failure models.